1. Technical Field
This disclosure relates to specimen-observation methods in a multi-beam microscope comprising both a charged-particle beam and an optical microscope. Examples of charged-particle beam microscopes include focused-ion beam microscopes (FIB), or scanning-electron microscopes (SEM) or combinations of the same (for example, a combined SEM/FIB), or a transmission electron microscope (TEM). This disclosure relates, in particular, to methods for generating optical and charged particle images from the same field of view while the sample is contained within a charged-particle beam microscope chamber.
2. Background
Life science researchers have traditionally relied on optical microscopy to examine tissue and cell samples and on TEM when needing to resolve subcellular details. Recent hardware improvements now enable SEMs to offer TEM-like images with resolution approximately 3-5 nm. Because of their larger chambers and reduced cost compared to TEM, SEMs offer an ideal platform for integration of combined imaging modalities. The ability to simultaneously image the same area of a sample with both electrons and photons enables real-time correlated imaging, avoids the need to generate entire image maps over large areas with optical microscopy, reduces the chance of sample damage during transfer between different microscopes, and saves analysis time.
Multi-beam charged-particle microscopes are commercially available. These instruments have a vacuum chamber that contains two or more charged particle imaging beams. The charged particle beams can include, but are not limited to, electron and ion beams such as in a scanning electron microscope (SEM), a focused ion beam microscope (FIB), and an SEM/FIB (also called a dual beam SEM/FIB or FIB/SEM) which has both a scanned ion beam and a scanned electron beam. FIBs typically use a gallium liquid metal ion source, but recent advances in imaging technology (such as the Orion He ion microscope (HIM) manufactured by Carl Zeiss Nano Technology Systems GmbH, Oberkochen, Germany) make possible the use of other ion beams for imaging purposes. Unless otherwise stated, a reference to imaging by an SEM or a charged-particle beam microscope should be taken to also refer to imaging by multi-beam ion and electron charged-particle beams, including ions of a variety of species such as the HIM or those delivered with a mass-filtered column technology, which offers sources based on Si, Cr, Fe, Co, Ni, Ge, In, Sn, Au, Mn, Pb, etc.
Since the images produced by a charged particle beam microscope and the optical microscope have widely varying resolutions and are formed based on different signals and contrast mechanisms, the image appearances and qualities are exceedingly different from each other. Consequently, it has been difficult and time-consuming to retrieve the observation field-of-view of the electron microscope on the basis of the specimen image acquired using the optical microscope. What is needed is a way to coordinate the images produced by the charged-particle beam and the optical illumination.